Delphi: A Democratic and Cost-Effective Method of Consensus Generation in Transplantation.

The Thrombotic Microangiopathy Banff Working Group (TMA-BWG) was formed in 2015 to survey current practices and develop minimum diagnostic criteria (MDC) for renal transplant TMA (Tx-TMA). To generate consensus among pathologists and nephrologists, the TMA BWG designed a 3-Phase study. Phase I of the study is presented here. Using the Delphi methodology, 23 panelists with >3 years of diagnostic experience with Tx-TMA pathology listed their MDC suggesting light, immunofluorescence, and electron microscopy lesions, clinical and laboratory information, and differential diagnoses. Nine rounds (R) of consensus resulted in MDC validated during two Rs using online evaluation of whole slide digital images of 37 biopsies (28 TMA, 9 non-TMA). Starting with 338 criteria the process resulted in 24 criteria and 8 differential diagnoses including 18 pathologic, 2 clinical, and 4 laboratory criteria. Results show that 3/4 of the panelists agreed on the diagnosis of 3/4 of cases. The process also allowed definition refinement for 4 light and 4 electron microscopy lesions. For the first time in Banff classification, the Delphi methodology was used to generate consensus. The study shows that Delphi is a democratic and cost-effective method allowing rapid consensus generation among numerous physicians dealing with large number of criteria in transplantation.

Thrombotic Microangiopathy in the Renal Allograft: Results of the TMA Banff Working Group Consensus on Pathologic Diagnostic Criteria.

The Banff community summoned the TMA Banff Working Group to develop minimum diagnostic criteria (MDC) and recommendations for renal transplant TMA (Tx-TMA) diagnosis, which currently lacks standardized criteria. Using the Delphi method for consensus generation, 23 nephropathologists (panelists) with >3 years of diagnostic experience with Tx-TMA were asked to list light, immunofluorescence, and electron microscopic, clinical and laboratory criteria and differential diagnoses for Tx-TMA. Delphi was modified to include 2 validations rounds with histological evaluation of whole slide images of 37 transplant biopsies (28 TMA and 9 non-TMA). Starting with 338 criteria in R1, MDC were narrowed down to 24 in R8 generating 18 pathological, 2 clinical, 4 laboratory criteria, and 8 differential diagnoses. The panelists reached a good level of agreement (70%) on 76% of the validated cases. For the first time in Banff classification, Delphi was used to reach consensus on MDC for Tx-TMA. Phase I of the study (pathology phase) will be used as a model for Phase II (nephrology phase) for consensus regarding clinical and laboratory criteria. Eventually in Phase III (consensus of the consensus groups) and the final MDC for Tx-TMA will be reported to the transplantation community.

Corrigendum: Delphi: A Democratic and Cost-Effective Method of Consensus Generation in Transplantation.

[This corrects the article DOI: 10.3389/ti.2023.11589.].

Corrigendum: Thrombotic Microangiopathy in the Renal Allograft: Results of the TMA Banff Working Group Consensus on Pathologic Diagnostic Criteria.

[This corrects the article DOI: 10.3389/ti.2023.11590.].

A psychiatric disease-related circular RNA controls synaptic gene expression and cognition.

Although circular RNAs (circRNAs) are enriched in the mammalian brain, very little is known about their potential involvement in brain function and psychiatric disease. Here, we show that circHomer1a, a neuronal-enriched circRNA abundantly expressed in the frontal cortex, derived from Homer protein homolog 1 (HOMER1), is significantly reduced in both the prefrontal cortex (PFC) and induced pluripotent stem cell-derived neuronal cultures from patients with schizophrenia (SCZ) and bipolar disorder (BD). Moreover, alterations in circHomer1a were positively associated with the age of onset of SCZ in both the dorsolateral prefrontal cortex (DLPFC) and orbitofrontal cortex (OFC). No correlations between the age of onset of SCZ and linear HOMER1 mRNA were observed, whose expression was mostly unaltered in BD and SCZ postmortem brain. Using in vivo circRNA-specific knockdown of circHomer1a in mouse PFC, we show that it modulates the expression of numerous alternative mRNA transcripts from genes involved in synaptic plasticity and psychiatric disease. Intriguingly, in vivo circHomer1a knockdown in mouse OFC resulted in specific deficits in OFC-mediated cognitive flexibility. Lastly, we demonstrate that the neuronal RNA-binding protein HuD binds to circHomer1a and can influence its synaptic expression in the frontal cortex. Collectively, our data uncover a novel psychiatric disease-associated circRNA that regulates synaptic gene expression and cognitive flexibility.

Transcriptomic changes due to early, chronic intermittent alcohol exposure during forebrain development implicate WNT signaling, cell-type specification, and cortical regionalization as primary determinants of fetal alcohol syndrome.

BACKGROUND: Fetal alcohol syndrome (FAS) due to gestational alcohol exposure represents one of the most common causes of nonheritable lifelong disability worldwide. In vitro and in vivo models have successfully recapitulated multiple facets of the disorder, including morphological and behavioral deficits, but far less is understood regarding the molecular and genetic mechanisms underlying FAS. METHODS: In this study, we utilized an in vitro human pluripotent stem cell-based (hPSC) model of corticogenesis to probe the effects of early, chronic intermittent alcohol exposure on the transcriptome of first trimester-equivalent cortical neurons. RESULTS: We used RNA sequencing of developing hPSC-derived neurons treated for 50 days with 50 mM ethanol and identified a relatively small number of biological pathways significantly altered by alcohol exposure. These included cell-type specification, axon guidance, synaptic function, and regional patterning, with a notable upregulation of WNT signaling-associated transcripts observed in alcohol-exposed cultures relative to alcohol-naïve controls. Importantly, this effect paralleled a shift in gene expression of transcripts associated with regional patterning, such that caudal forebrain-related transcripts were upregulated at the expense of more anterior ones. Results from H9 embryonic stem cells were largely replicated in an induced pluripotent stem cell line (IMR90-4), indicating that these patterning alterations are not cell line-specific. CONCLUSIONS: We found that a major effect of chronic intermittent alcohol on the developing cerebral cortex is an overall imbalance in regionalization, with enrichment of gene expression related to the production of posterodorsal progenitors and a diminution of anteroventral progenitors. This finding parallels behavioral and morphological phenotypes observed in animal models of high-dose prenatal alcohol exposure, as well as patients with FAS.

Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition.

Human parietal epithelial cells (PECs) are progenitor cells that sustain podocyte homeostasis. We hypothesized that the lack of apolipoprotein (APO) L1 ensures the PEC phenotype, but its induction initiates PEC transition (expression of podocyte markers). APOL1 expression and down-regulation of miR193a coincided with the expression of podocyte markers during the transition. The induction of APOL1 also stimulated transition markers in human embryonic kidney cells (cells with undetectable APOL1 protein expression). APOL1 silencing in PECs up-regulated miR193a expression, suggesting the possibility of a reciprocal feedback relationship between APOL1 and miR193a. HIV, interferon-γ, and vitamin D receptor agonist down-regulated miR193a expression and induced APOL1 expression along with transition markers in PECs. Luciferase assay suggested a putative interaction between miR193a and APOL1. Since silencing of APOL1 attenuated HIV-, vitamin D receptor agonist-, miR193a inhibitor-, and interferon-γ-induced expression of transition markers, APOL1 appears to be a critical functional constituent of the miR193a- APOL1 axis in PECs. This notion was confirmed by further enhanced expression of PEC markers in APOL1 mRNA-silenced PECs. In vivo studies, glomeruli in patients with HIV, and HIV/APOL1 transgenic mice had foci of PECs expressing synaptopodin, a transition marker. APOL1 likely regulates PEC molecular phenotype through modulation of miR193a expression, and APOL1 and miR193a share a reciprocal feedback relationship.

Genetic inactivation of synaptosomal-associated protein 25 (SNAP-25) in adult hippocampal neural progenitors impairs pattern discrimination learning but not survival or structural maturation of newborn dentate granule cells.

Adult neurogenesis is necessary for proper cognition and behavior, however, the mechanisms that underlie the integration and maturation of newborn neurons into the pre-existing hippocampal circuit are not entirely known. In this study, we sought to determine the role of action potential (AP)-dependent synaptic transmission by adult-generated dentate granule cells (DGCs) in their survival and function within the existing circuitry. We used a triple transgenic mouse (NestinCreERT2 :Snap25fl/fl : tdTomato) to inducibly inactivate AP-dependent synaptic transmission within adult hippocampal progenitors and their progeny. Behavioral testing in a hippocampal-dependent A/B contextual fear-discrimination task revealed impaired discrimination learning in mice harboring SNAP-25-deficient adult-generated dentate granule cells (DGCs). Despite poor performance on this neurogenesis-dependent task, the production and survival of newborn DGCs was quantitatively unaltered in tamoxifen-treated NestinCreERT2 :Snap25fl/fl : tdTomato SNAP compared to tamoxifen-treated NestinCreERT2 :Snap25wt/wt : tdTomato control mice. Although SNAP-25-deficient adult DGCs displayed a small but statistically significant enhancement in proximal dendritic branching, their overall dendritic length and distal branching complexity was unchanged. SNAP-25-deficient newborn DGCs also displayed robust efferent mossy fiber output to CA3, with normal linear density of large mossy fiber terminals (LMTs). These studies suggest that AP-dependent neurotransmitter release by newborn DGCs is not essential for their survival or rudimentary structural maturation within the adult hippocampus.

HIV Promotes NLRP3 Inflammasome Complex Activation in Murine HIV-Associated Nephropathy.

Dysregulated growth and loss of podocytes are important features of HIV-associated nephropathy. Recently, HIV was reported to induce a new type of programed cell death, pyroptosis, in T lymphocytes through induction of Nod-like receptor protein 3 (NLRP3) inflammasome complexes. We evaluated the role of HIV in podocyte NLRP3 inflammasome formation both in vivo and in vitro. Renal cortical sections of HIV-transgenic mice (Tg26) displayed increased expression of NLRP3, ASC (a CARD protein), caspase-1, and IL-1ß proteins, confirming NLRP3 inflammasome complex formation in podocytes of Tg26 mice. Renal tissues of Tg26 mice also displayed enhanced mRNA levels and protein expressions of inflammasome markers (NLRP3, ASC, and caspase-1, and IL-1ß). Serum of Tg26 mice also showed elevated concentrations of IL-1ß cytokine compared with FVBN mice. HIV induced pyroptosis in a dose- and time-dependent manner within podocytes, a phenotype of inflammasome activation. Caspase-1 inhibitor not only attenuated podocyte expression of caspase-1 and IL-1ß but also provided protection against pyroptosis, suggesting that HIV-induced podocyte injury was mediated by caspase-1 activation. Interestingly, HIV-induced podocyte pyroptosis could be partially inhibited by Tempol (a superoxide dismutase-mimetic agent) and by glyburide (an inhibitor of potassium efflux). These findings suggest that generation of reactive oxygen species and potassium efflux contribute to HIV-induced pyroptosis and NLRP3 inflammasome activation in podocytes.

Effects of Ethanol on Cellular Composition and Network Excitability of Human Pluripotent Stem Cell-Derived Neurons.

BACKGROUND: Prenatal alcohol exposure (PAE) in animal models results in excitatory-inhibitory (E/I) imbalance in neocortex due to alterations in the GABAergic interneuron (IN) differentiation and migration. Thus, E/I imbalance is a potential cause for intellectual disability in individuals with fetal alcohol spectrum disorder (FASD), but whether ethanol (EtOH) changes glutamatergic and GABAergic IN specification during human development remains unknown. Here, we created a human cellular model of PAE/FASD and tested the hypothesis that EtOH exposure during differentiation of human pluripotent stem cell-derived neurons (hPSNs) would cause the aberrant production of glutamatergic and GABAergic neurons, resulting in E/I imbalance. METHODS: We applied 50 mM EtOH daily to differentiating hPSNs for 50 days to model chronic first-trimester exposure. We used quantitative polymerase chain reaction, immunocytochemical, and electrophysiological analysis to examine the effects of EtOH on hPSN specification and functional E/I balance. RESULTS: We found that EtOH did not alter neural induction nor general forebrain patterning and had no effect on the expression of markers of excitatory cortical pyramidal neurons. In contrast, our data revealed highly significant changes to levels of transcripts involved with IN precursor development (e.g., GSX2, DLX1/2/5/6, NR2F2) as well as mature IN specification (e.g., SST, NPY). Interestingly, EtOH did not affect the number of GABAergic neurons generated nor the frequency or amplitude of miniature excitatory and inhibitory postsynaptic currents. CONCLUSIONS: Similar to in vivo rodent studies, EtOH significantly and specifically altered the expression of genes involved with IN specification from hPSNs, but did not cause imbalances of synaptic excitation-inhibition. Thus, our findings corroborate previous studies pointing to aberrant neuronal differentiation as an underlying mechanism of intellectual disability in FASD. However, in contrast to rodent binge models, our chronic exposure model suggests possible compensatory mechanisms that may cause more subtle defects of network processing rather than gross alterations in total E/I balance.

Curtailing endothelial TGF-ß signaling is sufficient to reduce endothelial-mesenchymal transition and fibrosis in CKD.

Excessive TGF-ß signaling in epithelial cells, pericytes, or fibroblasts has been implicated in CKD. This list has recently been joined by endothelial cells (ECs) undergoing mesenchymal transition. Although several studies focused on the effects of ablating epithelial or fibroblast TGF-ß signaling on development of fibrosis, there is a lack of information on ablating TGF-ß signaling in the endothelium because this ablation causes embryonic lethality. We generated endothelium-specific heterozygous TGF-ß receptor knockout (TßRII(endo+/-)) mice to explore whether curtailed TGF-ß signaling significantly modifies nephrosclerosis. These mice developed normally, but showed enhanced angiogenic potential compared with TßRII(endo+/+) mice under basal conditions. After induction of folic acid nephropathy or unilateral ureteral obstruction, TßRII(endo+/-) mice exhibited less tubulointerstitial fibrosis, enhanced preservation of renal microvasculature, improvement in renal blood flow, and less tissue hypoxia than TßRII(endo+/+) counterparts. In addition, partial deletion of TßRII in the endothelium reduced endothelial-to-mesenchymal transition (EndoMT). TGF-ß-induced canonical Smad2 signaling was reduced in TßRII(+/-) ECs; however, activin receptor-like kinase 1 (ALK1)-mediated Smad1/5 phosphorylation in TßRII(+/-) ECs remained unaffected. Furthermore, the S-endoglin/L-endoglin mRNA expression ratio was significantly lower in TßRII(+/-) ECs compared with TßRII(+/+) ECs. These observations support the hypothesis that EndoMT contributes to renal fibrosis and curtailing endothelial TGF-ß signals favors Smad1/5 proangiogenic programs and dictates increased angiogenic responses. Our data implicate endothelial TGF-ß signaling and EndoMT in regulating angiogenic and fibrotic responses to injury.

AT1R blockade in adverse milieus: role of SMRT and corepressor complexes.

ANG II type 1 receptor blockade (AT1R-BLK) is used extensively to slow down the progression of proteinuric kidney diseases. We hypothesized that AT1R-BLK provides podocyte protection through regulation of silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and vitamin D receptor (VDR) expression under adverse milieus such as high glucose and human immunodeficiency virus infection. Both AT1R-BLK and VDR agonists (VDAs) stimulated VDR complex formation that differed not only in their composition but also in their functionality. AT1R-BLK-induced VDR complexes contained predominantly unliganded VDR, SMRT, and phosphorylated histone deacetylase 3, whereas VDA-VDR complexes were constituted by liganded VDR and CREB-binding protein/p300. AT1R-BLK-induced complexes attenuated podocyte acetyl-histone 3 levels as well as cytochrome P-450 family 24A1 expression, thus indicating their deacetylating and repressive properties. On the other hand, VDA-VDR complexes not only increased podocyte acetyl-histone 3 levels but also enhanced cytochrome P-450 family 24A1 expression, thus suggesting their acetylating and gene activation properties. AT1R-BLK- induced podocyte SMRT inhibited expression of the proapoptotic gene BAX through downregulation of Wip1 and phosphorylation of checkpoint kinase 2 in high-glucose milieu. Since SMRT-depleted podocytes lacked AT1R-BLK-mediated protection against DNA damage, it appears that SMRT is necessary for DNA repairs during AT1R-BLK. We conclude that AT1R-BLK provides podocyte protection in adverse milieus predominantly through SMRT expression and partly through unliganded VDR expression in 1,25(OH)2D-deficient states; on the other hand, AT1R-BLK contributes to liganded VDR expression in 1,25(OH)2D-sufficient states.

VLK drives extracellular phosphorylation of EphB2 to govern the EphB2-NMDAR interaction and injury-induced pain.

Phosphorylation of hundreds of protein extracellular domains is mediated by two kinase families, yet the significance of these kinases is underexplored. Here, we find that the presynaptic release of the tyrosine directed-ectokinase, Vertebrate Lonesome Kinase (VLK/Pkdcc), is necessary and sufficient for the direct extracellular interaction between EphB2 and GluN1 at synapses, for phosphorylation of the ectodomain of EphB2, and for injury-induced pain. Pkdcc is an essential gene in the nervous system, and VLK is found in synaptic vesicles, and is released from neurons in a SNARE-dependent fashion. VLK is expressed by nociceptive sensory neurons where presynaptic sensory neuron-specific knockout renders mice impervious to post-surgical pain, without changing proprioception. VLK defines an extracellular mechanism that regulates protein-protein interaction and non-opioid-dependent pain in response to injury.

mTOR plays a critical role in p53-induced oxidative kidney cell injury in HIVAN.

Oxidative stress has been implicated to contribute to HIV-induced kidney cell injury; however, the role of p53, a modulator of oxidative stress, has not been evaluated in the development of HIV-associated nephropathy (HIVAN). We hypothesized that mammalian target of rapamycin (mTOR) may be critical for the induction of p53-mediated oxidative kidney cell injury in HIVAN. To test our hypothesis, we evaluated the effect of an mTOR inhibitor, rapamycin, on kidney cell p53 expression, downstream signaling, and kidney cell injury in both in vivo and in vitro studies. Inhibition of the mTOR pathway resulted in downregulation of renal tissue p53 expression, associated downstream signaling, and decreased number of sclerosed glomeruli, tubular microcysts, and apoptosed and 8-hydroxy deoxyguanosine (8-OHdG)-positive (+ve) cells in Tg26 mice. mTOR inhibition not only attenuated kidney cell expression of p66ShcA and phospho-p66ShcA but also reactivated the redox-sensitive stress response program in the form of enhanced expression of manganese superoxide dismutase (MnSOD) and catalase. In in vitro studies, the mTOR inhibitor also provided protection against HIV-induced podocyte apoptosis. Moreover, mTOR inhibition downregulated HIV-induced podocyte (HP/HIV) p53 expression. Since HP/HIV silenced for mTOR displayed a lack of expression of p53 as well as attenuated podocyte apoptosis, this suggests that mTOR is critical for kidney cell p53 activation and associated oxidative kidney cell injury in the HIV milieu.

Parietal Epithelial Cell Behavior and Its Modulation by microRNA-193a.

Glomerular parietal epithelial cells (PECs) have been increasingly recognized to have crucial functions. Lineage tracking in animal models showed the expression of a podocyte phenotype by PECs during normal glomerular growth and after acute podocyte injury, suggesting a reparative role of PECs. Conversely, activated PECs are speculated to be pathogenic and comprise extracapillary proliferation in focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CrescGN). The reparative and pathogenic roles of PECs seem to represent two sides of PEC behavior directed by the local milieu and mediators. Recent studies suggest microRNA-193a (miR193a) is involved in the pathogenesis of FSGS and CrescGN. In a mouse model of primary FSGS, the induction of miR193a caused the downregulation of Wilms' tumor protein, leading to the dedifferentiation of podocytes. On the other hand, the inhibition of miR193a resulted in reduced crescent lesions in a mouse model of CrescGN. Interestingly, in vitro studies report that the downregulation of miR193a induces trans-differentiation of PECs into a podocyte phenotype. This narrative review highlights the critical role of PEC behavior in health and during disease and its modulation by miR193a.

Transsynaptic Signaling of Ephs in Synaptic Development, Plasticity, and Disease.

Synapse formation between neurons is critical for proper circuit and brain function. Prior to activity-dependent refinement of connections between neurons, activity-independent cues regulate the contact and recognition of potential synaptic partners. Formation of a synapse results in molecular recognition events that initiate the process of synaptogenesis. Synaptogenesis requires contact between axon and dendrite, selection of correct and rejection of incorrect partners, and recruitment of appropriate pre- and postsynaptic proteins needed for the establishment of functional synaptic contact. Key regulators of these events are families of transsynaptic proteins, where one protein is found on the presynaptic neuron and the other is found on the postsynaptic neuron. Of these families, the EphBs and ephrin-Bs are required during each phase of synaptic development from target selection, recruitment of synaptic proteins, and formation of spines to regulation of synaptic plasticity at glutamatergic spine synapses in the mature brain. These roles also place EphBs and ephrin-Bs as important regulators of human neurological diseases. This review will focus on the role of EphBs and ephrin-Bs at synapses.

Distribution and inflammatory cell response to intracranial delivery of radioluminescent Y2(SiO4)O:Ce particles.

Due to increasing advances in their manufacture and functionalization, nanoparticle-based systems have become a popular tool for in vivo drug delivery and biodetection. Recently, scintillating nanoparticles such as yttrium orthosilicate doped with cerium (Y2(SiO4)O:Ce) have come under study for their potential utility in optogenetic applications, as they emit photons upon low levels of stimulation from remote x-ray sources. The utility of such nanoparticles in vivo is hampered by rapid clearance from circulation by the mononuclear phagocytic system, which heavily restricts nanoparticle accumulation at target tissues. Local transcranial injection of nanoparticles may deliver scintillating nanoparticles to highly specific brain regions by circumventing the blood-brain barrier and avoiding phagocytic clearance. Few studies to date have examined the distribution and response to nanoparticles following localized delivery to cerebral cortex, a crucial step in understanding the therapeutic potential of nanoparticle-based biodetection in the brain. Following the synthesis and surface modification of these nanoparticles, two doses (1 and 3 mg/ml) were introduced into mouse secondary motor cortex (M2). This region was chosen as the site for RLP delivery, as it represents a common target for optogenetic manipulations of mouse behavior, and RLPs could eventually serve as an injectable x-ray inducible light delivery system. The spread of particles through the target tissue was assessed 24 hours, 72 hours, and 9 days post-injection. Y2(SiO4)O:Ce nanoparticles were found to be detectable in the brain for up to 9 days, initially diffusing through the tissue until 72 hours before achieving partial clearance by the final endpoint. Small transient increases in the presence of IBA-1+ microglia and GFAP+ astrocytic cell populations were detected near nanoparticle injection sites of both doses tested 24 hours after surgery. Taken together, these data provide evidence that Y2(SiO4)O:Ce nanoparticles coated with BSA can be injected directly into mouse cortex in vivo, where they persist for days and are broadly tolerated, such that they may be potentially utilized for remote x-ray activated stimulation and photon emission for optogenetic experiments in the near future.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) in cellular processes: Focus on hnRNP E1's multifunctional regulatory roles.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) comprise a family of RNA-binding proteins. The complexity and diversity associated with the hnRNPs render them multifunctional, involved not only in processing heterogeneous nuclear RNAs (hnRNAs) into mature mRNAs, but also acting as trans-factors in regulating gene expression. Heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1), a subgroup of hnRNPs, is a KH-triple repeat containing RNA-binding protein. It is encoded by an intronless gene arising from hnRNP E2 through a retrotransposition event. hnRNP E1 is ubiquitously expressed and functions in regulating major steps of gene expression, including pre-mRNA processing, mRNA stability, and translation. Given its wide-ranging functions in the nucleus and cytoplasm and interaction with multiple proteins, we propose a post-transcriptional regulon model that explains hnRNP E1's widespread functional diversity.

Adverse host factors exacerbate occult HIV-associated nephropathy.

In the present study, we hypothesized that HIV-1-induced occult HIV-associated nephropathy (HIVAN) would become apparent in the presence of adverse host factors. To test our hypothesis, Vpr mice (which display doxycycline-dependent Vpr expression in podocytes) with two, three, and four copies of the angiotensinogen (Agt) gene (Vpr-Agt-2, Vpr-Agt-3, and Vpr-Agt-4) were administered doxycycline for 3 weeks (to develop clinically occult HIVAN) followed by doxycycline-free water during the next 3 weeks. Subsequently, renal biomarkers were measured, and kidneys were harvested for renal histology. Vpr-Agt-2 developed neither proteinuria nor elevated blood pressure, and displayed minimal glomerular and tubular lesions only, without any microcyst formation. Vpr-Agt-3 showed mild glomerular and tubular lesions and microcyst formation, whereas Vpr-Agt-4 showed moderate proteinuria, hypertension, glomerular sclerosis, tubular dilation, microcysts, and expression of epithelial mesenchymal transition markers. Vpr-Agt-4 not only displayed enhanced renal tissue expression of Agt, renin, and angiotensin-converting enzyme, but also had higher renal tissue concentrations of angiotensin II. Moreover, renal cells in Vpr-Agt-4 showed enhanced expression of transforming growth factor-ß, connective tissue growth factor, and vascular endothelial growth factor. These findings indicate that adverse host factors, such as the activation of the renin-angiotensin system, promote the progression of occult HIVAN to apparent HIVAN.